The goal of this section is to explore the function of matrix proteins made by cells in skeletal tissues. Our current strategy is to focus on a single category of extracellular matrix (ECM) proteins known as small leucine rich proteoglycans (SLRPs). They were selected based on their high level of expression in skeletal tissues. SLRPs are part of a growing family currently composed of 13 members that are divided into classes I, II and III based on their amino acid sequences and intron-exon structures. At least 9 of the 13 SLRPs are expressed in skeletal tissues, but their precise function in these tissues remains unknown. Our working hypothesis is that SLRPs have important roles in controlling the structure and function of bones and teeth. We have tested this hypothesis by creating mice deficient in one or more SLRPs and examining the resulting phenotypes at the structural, cellular and molecular levels. mRNA PROFILING TO STUDY BIGLYCAN FUNCTION IN SKELETAL CELLS (33% effort) Biglycan is one of the founding members of the SLRP (small leucine rich proteoglycans) family. Previous studies from the lab showed that mice deficient in biglycan develop early onset osteoporosis caused by defects in osteoblast function and, subsequently, bone formation. To identify factors that play a role in controlling biglycan-dependent osteoblast differentiation, a series of microarray experiments was performed. In this study the global gene expression patterns in normal and biglycan-deficient calvarial cells treated with or without BMP-4 were determined using Affymetrix chip technology. Extensive bioinformatics analysis identified multiple hierarchical gene clusters whose expression was altered in ways that were dependent on biglycan, BMP-4 or both. A wide variety of genes whose functions are known to be related to proliferation, differentiation and survival were found to be differentially expressed. The array analysis also showed changes in the expression of mRNA?s related to inflammation and tumor suppression leading us to consider additional functions for biglycan in bone that can now be tested. In summary, the array analysis provided a plentiful dowry of new information about the coordinated mRNA expression that is dependent on the presence of biglycan. This information will, in turn, be used to devise new theoretical concepts about molecular cross-talk between biglycan function and skeletal cell signaling. USING BIGLYCAN AND DECORIN DEFICIENT MICE TO STUDY FUNCTIONAL COMPENSATION BETWEEN SLRPS (34% effort) Several types of analyses showed that when biglycan is absent there was an induction of the related SLRP, decorin. In order to determine whether decorin could partially compensate for the absence of biglycan, we created mice deficient in both proteoglycans. The doubly mutant male mice had earlier and more severe osteopenia compared to biglycan- or decorin-deficient mice. To determine the cellular basis for this skeletal defect, bone marrow stromal cells (BMSCs) were isolated from the marrow of adult biglycan/decorin-deficient mice and shown to have: 1) markedly increased proliferation 2) increased TGF-beta signaling and 3) premature apoptosis (programmed cell death) compared to normal cells. To understand the mechanistic foundation for these observations, we examined the localization of TGF-beta in normal and mutant cells. In the absence of both biglycan and decorin, TGF-beta inefficiently bound to the extracellular matrix and in its free form could over-activate TGF-beta-induced downstream signaling pathways. The outcome of this uncontrolled growth factor stimulation was premature cell death of the BMSCs, leading to decreased osteogenesis and, ultimately, osteopenia. It is important to note that, in the single biglycan-KO mice, there was a clear increase and redistribution of decorin accumulation that appeared to alter the matrix microenvironment such that growth factor activity was limited. In the absence of both proteoglycans, this type of compensation could not occur and caused instead uncontrolled growth factor stimulation and accelerated cell demise. These data suggest that the mechanisms causing osteopenia in the biglycan KO mice are distinct from those that caused osteopenia in the double biglycan/decorin KO mice. This concept further points to the conclusion that removal of one or more SLRP?s can have unique molecular ramifications that need to be dissected separately for each SLRP KO mouse line. CHARACTERIZATION OF BIGLYCAN AND FIBROMODULIN DEFICIENT MICE AS MODELS OF TEMPOROMANDIBULAR JOINT-OSTEOARTHRITIS (33% effort) Biglycan (Class I) and the more distantly related SLRP fibromodulin (Class II) are highly expressed in the disc and articular cartilage of the temporomandibular joint (TMJ). We theorized that the combined absence of these two SLRPs would result in accelerated osteoarthritis in the temporomandibular joint (TMJ). Histological sections of TMJ from 3, 6, 9 and 18 month-old wild-type (wt) and fibromodulin/biglycan double-deficient (DKO) mice were compared. At 3 months of age, both wt and DKO animals presented early signs of cartilage degeneration, visible as small acellular areas under the articular surfaces and superficial waving. From 6 months of age, DKOs developed accelerated osteoarthritis compared to wt. At 6 months, small vertical clefts in the condylar cartilage and partial disruption of the disk were visible in the DKOs. In addition, chondrocytes lost regular columnar organization and formed clusters. At 9 months, these differences were even more pronounced. At 18 months, extended cartilage erosion was visible in DKOs, when, by comparison, the thickness of the articular cartilage in wt controls was intact. Proliferating cell nuclear antigen (PCNA) staining to estimate proliferation levels was more pronounced in 3 month-old wt TMJ fibrocartilage compared to 3 month-old DKO TMJ fibrocartilage, suggesting that chondrocyte proliferation might be impaired in DKOs. We conclude from this work that the biglycan/fibromodulin double knock-out mouse constitutes a useful animal model to decipher the pathobiology of osteoarthritis in the TMJ. In summary, our studies show that SLRPs are critical for maintaining normal skeletal tissue architecture and that loss of SLRP function leads to defects in osteogenesis and osteoblast differentiation, as well as growth factor distribution and utilization. The SLRP-deficient mouse lines provide novel animal models with numerous skeletal defects. Our future goals will be to use the mouse lines to further understand the cellular and molecular events that cause the skeletal defects caused by the absence of one or more SLRP.